Gene therapy using genetically engineered cells and viruses has undergone impressive development over the past 40 years. Gene therapy techniques have been applied to diverse medical problems and have been used in over 350 clinical trials (Wu et al., Meth. Strat. Anesthes. (2001) 94:1119-1132).
Gene therapy has been attempted using various virus particles, including recombinant adeno-associated virus (rAAV) virions encoding therapeutic transgenes. Recombinant AAV vectors can be readily constructed using techniques well known in the art. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92/01070 and WO 93/03769; Lebkowski et al., Molec. Cell. Biol. (1988) 8:3988-3996; Vincent et al., Vaccines 90 (1990) (Cold Spring Harbor Laboratory Press); Carter, B. J. Current Opinion in Biotechnology (1992) 3:533-539; Muzyczka, N. Current Topics in Microbiol. and Immunol. (1992) 158:97-129; Kotin, R. M. Human Gene Therapy (1994) 5:793-801; Shelling and Smith, Gene Therapy (1994) 1:165-169; and Zhou et al., J. Exp. Med. (1994) 179:1867-1875. Although such AAV virion-mediated gene delivery has been shown to give rise to long term expression of transgenes in target cells, and long term therapeutic benefit in animal models, rAAV virus particles can generate an adverse immune response in human subjects. In addition, the packaging limit for AAV virions is approximately 5000 bases, which limits the size of the transgene construct that can be delivered using such virions.
Gene therapy vectors derived from other viruses have disadvantages as well. A number of retroviral-based gene delivery systems have been described. See, e.g., U.S. Pat. No. 5,219,740; Miller and Rosman, BioTechniques (1989) 7:980-990; Miller, A. D., Human Gene Therapy (1990) 1:5-14; Scarpa et al., Virology (1991) 180:849-852; Burns et al., Proc. Natl. Acad. Sci. USA (1993) 90:8033-8037; and Boris-Lawrie and Temin, Cur. Opin. Genet. Develop. (1993) 3:102-109. Retroviral virion-mediated gene therapy, however, can result in integration of the transgene into the chromosome of the target cell at a random location, which could create an undesirable mutation.
A number of adenovirus vectors have also been described. Unlike retroviruses which integrate into the host genome, adenoviruses persist extrachromosomally thus minimizing the risks associated with insertional mutagenesis (Haj-Ahmad and Graham, J. Virol. (1986) 57:267-274; Bett et al., J. Virol. (1993)67:5911-5921; Mittereder et al., Human Gene Therapy (1994) 5:717-729; Seth et al., J. Virol. (1994) 68:933-940; Barr et al., Gene Therapy (1994) 1:51-58; Berkner, K. L. BioTechniques (1988) 6:616-629; and Rich et al., Human Gene Therapy (1993) 4:461-476). Adenovirus virion-mediated gene therapy, however, is plagued by potentially serious immune response in humans.
As an alternative to viral vectors, plasmid DNA (pDNA) has also been used as a vector for gene delivery, as described in U.S. Pat. Nos. 6,413,942; 6,214,804; 5,580,859; 5,589,466; 5,763,270; and 5,693,622, the disclosures of which are hereby incorporated by reference in their entireties. Plasmids have an advantage viral vectors because the production of sufficient quantities of virions at sufficiently high titer is difficult and expensive. Plasmids will include the gene of interest operably linked to control elements that direct the expression of the protein product in vivo. Such control elements are well known in the art. Transgene expression is dependent on several features of the plasmid. The transcription of the transgene is controlled by the enhancer/promoter sequences, acting singly or in combination, incorporated into the sequence of the pDNA. Stability of the transcribed message is enhanced by the presence of a poly-adenylation signal at the 3′ end of the transcript, known as the polyA tail. Transcription termination sequences may also be of importance, as are other regulatory domains, such as internal ribosome entry sites (“IRES”), and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control sequences need always be present so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell.
Plasmid-mediated gene therapy, however, has typically only directed transient, rather than sustained, transgene expression in target cells.
The need exists for improved methods of gene therapy that are capable of directing sustained transgene expression without the adverse consequences and expense associated with virion-mediated gene therapy methods.